Diminished loading of the skeleton (i.e., disuse) precipitates a series of cellular events culminating in locally mediated bone loss. However, the signaling pathways by which disuse is transduced into osteoclastogenesis and subsequent bone resorption have not been identified. In our initial funding cycle, we generated data that substantiated our general hypothesis that disuse induces osteocyte hypoxia. In this competitive renewal, we progress to a more mechanistic understanding of how osteocyte hypoxia may directly mediate osteoclastic activity within a. physiologic context. Specifically, we hypothesize that normalization of disuse induced osteocyte osteopontin (OPN) expression will inhibit disuse induced intracortical remodeling. To examine this hypothesis, we will implement a multi-disciplinary approach utilizing complementary in vivo (avian ulna model of disuse osteopenia) and in vitro (MLO-Y4 osteocyte) models. In a series of Specific Aims, we will: 1) define the time course of osteocyte OPN expression, osteoclastic presence, intracortical and endocortical resorption volume in response to acute and chronic disuse, 2) define an intermittent in vivo mechanical loading regimen and an in vitro re-oxygenation regimen that normalizes osteocyte OPN expression in response to disuse or direct hypoxia, 3) define the period of time by which mechanical loading and re-oxygenation must be initiated in order to normalize osteocyte OPN expression in response to disuse or direct hypoxia, and 4) normalize osteocyte OPN expression during a critical window of time by superimposing daily mechanical loading upon chronic disuse. Without chemotaxant signals from osteocytes (i.e., OPN), we propose that osteoclasts (or their pre-cursors) will be less likely to initiate intracortical resorption. If successful, we anticipate that we will inhibit disuse induced intracortical resorption and thereby substantially diminish the tissue degradation and loss of bone strength associated with disuse. If so, these data would identify a novel pathway by which disuse induced osteoclastic activity is directly mediated. In our view, the therapeutic potential of this information is substantial as the ability to inhibit disuse induced intracortical resorption could counteract bone fragility and ancillary health issues arising from spinal cord injury, stroke, extended bedrest following major surgery, or fracture healing.